| Hepatocellular carcinoma(HCC)is the most common primary liver cancer,and the morbidity of HCC is ranked at the fifth,the mortality is the third.Drug/gene co-delivery system is a novel delivery methods.Due to their positive charge on the surface,polycation molecules combine with cell membrane.Meanwhile,because their surface can be modified and combine with gene static,their improve drug safety and accuracy by introducing functional groups to enhance their targetability.In this work,we performed galactosylated chitosan and fluorouracil acetic acid(GC-FUA)to prepare macromolecular prodrugs of galactosylated chitosan-5-fluorouracil acetic acid(GC-FUA),and introduce liver specific expression gene,micro RNA-122,to contract macromolecule prodrug/gene delivery system.Moreover,we explore their anti-tumor effect on hepatocellular carcinoma HepG2 cells in vitro.This paper is structured as follows:According to early laboratory research foundation,we synthesized macromolecular prodrugs of GC-FUA,and fabricated Nano system.We constructed GC-FUA /microRNA-122 co-deliveryNano-system through a combination of electrostatic miR-122 mimics and macromolecular prodrugs passed by the appropriate coupling.Agarose gel electrophoresis was used to detect combine of miR-122 and GC-FUA,UV spectrophotometry(UV)test was used to detect the drug load radio and gene loading radio of GC-FUA/mi R-122 Nano-system.Meanwhile,the nano-composite particle size and Zeta potential were tested.The results showed that we have successfully built GC-FUA/mi R-122co-delivery nano-system when the mass ratio of GC-FUA:miR-122 is equal to 1024:1,and nano-system is stable when GC-FUA:mi R-122 >64:1.What's more,we evaluated the biocompatibilities of co-delivery nano-system.CCK8 was used to detected cytotoxicity of normal liver cells(LO2)and vascular endothelial cells(ECs)after treating the GC.In this work,the protein adsorption test was carried out with BSA as a model protein to examine the stability in blood circulation and potential application of nano-system in vivo.In addition,the HR of copolypeptides was assessed by a hemolysis assay.An enhanced HR results in a higher level of broken RBCs.Results suggested that the nano-system exhibited low cytotoxicities and could be safely used as biocompatible carriers for delivering bioactive substances.To testify the co-delivery Nano system have a impact in liver cancer in vitro,we performed transfection with GFP labeledmiR-122 mimics in HepG2 cells to observe the uptake ratio of Nano-composite system.Moreover,CCK8 method was employed to detect proliferation of HepG2 cells after treated with the Nano co-delivery system.And FCM also was used to test fluorescence intensity of Nano-composite systems in HepG2 cells,as well as detect HepG2 cell apoptosis.Furthe rmore,cell wound healing test and transwell method were carried out to detecte migration and invasion in HepG2.To further verify that the Nano co-delivery system have a anti-tumor effect in HepG2 cell in vitro,westrn blot was performed to detect expression of Bcl-2,and adam17 of mi R-122 gene targers.Our results showed that GC-FUA/mi R-122 significantly inhibited HepG2 cell proliferation compared with free 5-Fu.Meanwhile,GC-FUA/miR-122 remarkedly induced HepG2 cell apoptosis,and inhibited migration and invasion of HepG2 cells.We observed that GC-FUA/miR-122 downrugulated expression of Bcl-2,Adam17 of miR-122 gene targets,as dertermined by western blot.Conclusion: we have been successfully constructed prodrug/gene co-delivery nano system.And nano-system exhibited low cytotoxicities and could be safely used as biocompatible carriers for delivering bioactive substances.Compared with free 5-Fu,nano-delivery system had a better inhibition of the HepG2 cells proliferation.Meanwhile,GC-FUA/miR-122 remarkedly induced HepG2 cell apoptosis,and inhibited migration and invasion of HepG2 cells.We observed that GC-FUA/mi R-122 downrugulated expression of Bcl-2,Adam17 of miR-122 gene targets,as dertermined by western blot. |
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